Polypeptides for inducing a protective immune response against staphylococcus aureus

ABSTRACT

The present invention features polypeptides comprising an amino acid sequence structurally related to SEQ ID NO: 1 or a fragment thereof,  S. aureus  AhpC-AhpF compositions, and uses of such polypeptides and compositions. SEQ ID NO: 1 has a full length  S. aureus  AhpC sequence. A derivative of SEQ ID NO: 1 containing an amino His-tag and three additional carboxylamino acids was found to produce a protective immune response against  S. aureus.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.11/795,538, filed Jul. 18, 2007, currently pending, which is a §371National Stage application of PCT/US06/01665, filed Jan. 17, 2006, whichclaims the benefit of U.S. Provisional Application No. 60/645,811 filedJan. 21, 2005, which is hereby incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “MRLIFD21722USDIV_SEQLIST_(—)9MARCH2010.TXT”, creation date ofMar. 9, 2010, and a size of 26 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The references cited throughout the present application are not admittedto be prior art to the claimed invention.

Staphylococcus aureus is a pathogen responsible for a wide range ofdiseases and conditions. Examples of diseases and conditions caused byS. aureus include bacteremia, infective endocarditis, folliculitis,furuncle, carbuncle, impetigo, bullous impetigo, cellulitis,botryomyosis, toxic shock syndrome, scalded skin syndrome, centralnervous system infections, infective and inflammatory eye disease,osteomyelitis and other infections of joints and bones, and respiratorytract infections. (The Staphylococci in Human Disease, Crossley andArcher (eds.), Churchill Livingstone Inc. 1997.)

Immunological based strategies can be employed to control S. aureusinfections and the spread of S. aureus. Immunological based strategiesinclude passive and active immunization. Passive immunization employsimmunoglobulins targeting S. aureus. Active immunization induces immuneresponses against S. aureus.

Potential S. aureus vaccines target S. aureus polysaccharides andpolypeptides. Targeting can be achieved using suitable S. aureuspolysaccharides or polypeptides as vaccine components. Examples ofpolysaccharides that may be employed as possible vaccine componentsinclude S. aureus type 5 and type 8 capsular polysaccharides.(Shinefield et al., N. Eng. J. Med. 346:491-496, 2002.) Examples ofpolypeptides that may be employed as possible vaccine components includecollagen adhesin, fibrinogen binding proteins, and clumping factor.(Mamo et al., FEMS Immunology and Medical Microbiology 10:47-54, 1994,Nilsson et al., J. Clin. Invest. 101:2640-2649, 1998, Josefsson et al.,The Journal of Infectious Diseases 184:1572-1580, 2001.)

Information concerning S. aureus polypeptide sequences has been obtainedfrom sequencing the S. aureus genome. (Kuroda et al., Lancet357:1225-1240, 2001, Baba et al., Lancet 359:1819-1827, 2000, Kunsch etal., European Patent Publication EP 0 786 519, published Jul. 30, 1997.)To some extent bioinformatics has been employed in efforts tocharacterize polypeptide sequences obtained from genome sequencing.(Kunsch et al., European Patent Publication EP 0 786 519, published Jul.30, 1997.)

Techniques such as those involving display technology and sera frominfected patients have been used in an effort to help identify genescoding for potential antigens. (Foster et al., International PublicationNumber WO 01/98499, published Dec. 27, 2001, Meinke et al.,International Publication Number WO 02/059148, published Aug. 1, 2002,Etz et al., PNAS 99:6573-6578, 2002.)

SUMMARY OF THE INVENTION

The present invention features polypeptides comprising an amino acidsequence structurally related to SEQ ID NO: 1 or a fragment thereof, S.aureus AhpC-AhpF compositions, and uses of such polypeptides andcompositions. SEQ ID NO: 1 has a full length S. aureus AhpC sequence. Aderivative of SEQ ID NO: 1 containing an amino His-tag and threeadditional carboxylamino acids was found to produce a protective immuneresponse against S. aureus.

Reference to “protective” immunity or immune response indicates adetectable level of protection against S. aureus infection. The level ofprotection can be assessed using animal models such as those describedherein.

Thus, a first aspect of the present invention describes a polypeptideimmunogen comprising an amino acid sequence at least 85% identical toSEQ ID NO: 1 or to a fragment of SEQ ID NO: 1, wherein the polypeptideprovides protective immunity against S. aureus and the polypeptideimmunogen is not the polypeptide of SEQ ID NO: 1. Reference to immunogenindicates the ability to provide protective immunity against S. aureus.

Reference to comprising an amino acid sequence at least 85% identical toSEQ ID NO: 1 indicates that a SEQ ID NO: 1 related region is present andadditional polypeptide regions may be present. Percent identity (alsoreferred to as percent identical) to a reference sequence is determinedby aligning the polypeptide sequence with the reference sequence anddetermining the number of identical amino acids in the correspondingregions. This number is divided by the total number of amino acids inthe reference sequence (e.g., SEQ ID NO: 1) and then multiplied by 100and rounded to the nearest whole number.

Another aspect of the present invention describes an immunogencomprising a polypeptide that provides protective immunity against S.aureus and one or more additional regions or moieties covalently joinedto the polypeptide at the carboxyl terminus or amino terminus, whereineach region or moiety is independently selected from a region or moietyhaving at least one of the following properties: enhances the immuneresponse, facilitates purification, or facilitates polypeptidestability.

Reference to “additional region or moiety” indicates a region or moietydifferent from a S. aureus AhpC region. The additional region or moietycan be, for example, an additional polypeptide region or a non-peptideregion.

Another aspect of the present invention describes a purified immunogenmade up of an AhpC-AhpF composition. The AhpC component comprises apolypeptide at least 85% identical to SEQ ID NO: 1. The AhpF componentcomprises a polypeptide at least 85% identical to SEQ ID NO: 3.Reference to purified indicates that the composition is present in anenvironment lacking one or more other polypeptides with which AhpC andAhpF is naturally associated and/or represents at least about 10% of thetotal protein present.

Preferably, the composition is substantially purified. A “substantiallypurified” AhpC and AhpF composition is present in an environment lackingall, or most, other polypeptides with which AhpC and AhpF polypeptide isnaturally associated.

Reference to “purified” or “substantially purified” does not require apolypeptide to undergo any purification and may include, for example, achemically synthesized polypeptide that has not been purified.

Another aspect of the present invention describes a composition able toinduce protective immunity against S. aureus in a patient. Thecomposition comprises a pharmaceutically acceptable carrier and animmunologically effective amount of an immunogen providing protectiveimmunity against S. aureus.

An immunologically effective amount is an amount sufficient to provideprotective immunity against S. aureus infection. The amount should besufficient to significantly prevent the likelihood or severity of a S.aureus infection.

Another aspect of the present invention describes a nucleic acidcomprising a recombinant gene encoding a polypeptide that providesprotective immunity against S. aureus. A recombinant gene containsrecombinant nucleic acid encoding a polypeptide along with regulatoryelements for proper transcription and processing (which may includetranslational and post translational elements). The recombinant gene canexist independent of a host genome or can be part of a host genome.

A recombinant nucleic acid is nucleic acid that by virtue of itssequence and/or form does not occur in nature. Examples of recombinantnucleic acid include purified nucleic acid, two or more nucleic acidregions combined together that provide a different nucleic acid thanfound in nature, and the absence of one or more nucleic acid regions(e.g., upstream or downstream regions) that are naturally associatedwith each other.

Another aspect of the present invention describes a recombinant cell.The cell comprises a recombinant gene encoding a polypeptide thatprovides protective immunity against S. aureus.

Another aspect of the present invention describes a method of making apolypeptide that provides protective immunity against S. aureus. Themethod involves growing a recombinant cell containing recombinantnucleic acid encoding the polypeptide and purifying the polypeptide.

Another aspect of the present invention describes a polypeptide thatprovides protective immunity against S. aureus made by a processcomprising the steps of growing a recombinant cell containingrecombinant nucleic acid encoding the polypeptide in a host andpurifying the polypeptide. Different host cells can be employed.

Another aspect of the present invention describes an isolated AhpC-AhpFbinding protein. The binding protein comprises an antibody variableregion that binds to an AhpC-AhpF complex.

Reference to “isolated” indicates a different form than found in nature.The different form can be, for example, a different purity than found innature and/or a structure that is not found in nature. A structure notfound in nature includes recombinant structures where different regionsare combined together, for example, humanized antibodies where one ormore murine complementary determining regions (CDR) is inserted onto ahuman framework scaffold, hybrid antibodies where one or more CDR froman antibody binding protein is inserted into a different frameworkscaffold, and antibodies derived from natural human sequences wheregenes coding light and heavy variable domains were randomly combinedtogether.

The isolated protein is preferably substantially free of serum proteins.A protein substantially free of serum proteins is present in anenvironment lacking most or all serum proteins.

Another aspect of the present invention describes a method of treating apatient against S. aureus infection. The method comprises the step ofadministering to the patient one or more of the following:

(a) an immunologically effective amount of an immunogen comprising anamino acid sequence at least 85% identical to SEQ ID NO: 1 or a fragmentof SEQ ID NO: 1, wherein the polypeptide provides protective immunityagainst S. aureus:

(b) an immunogenic composition comprising a polypeptide at least 85%identical to SEQ ID NO: 1 and a polypeptide at least 85% identical toSEQ ID NO: 3; or

(c) an effective amount of an AhpC-AhpF binding protein.

Unless particular terms are mutually exclusive, reference to “or”indicates either or both possibilities. Occasionally phrases such as“and/or” are used to highlight either or both possibilities.

Reference to open-ended terms such as “comprises” allows for additionalelements or steps. Occasionally phrases such as “one or more” are usedwith or without open-ended terms to highlight the possibility ofadditional elements or steps.

Unless explicitly stated reference to terms such as “a” or “an” is notlimited to one. For example, “a cell” does not exclude “cells”.Occasionally phrases such as one or more are used to highlight thepossible presence of a plurality.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of SEQ ID NO: 1 and SEQ IDNO: 2. The entire sequence is SEQ ID NO: 2. The portion shown in bold isSEQ ID NO: 1. The underlined regions are an amino His-tag region andadditional amino acids at the carboxyl end.

FIG. 2 illustrates a sequence comparison between AhpC sequences from S.aureus (SEQ ID NO: 1) and S. epidermidis (SEQ ID NO: 6, GenBankAccession No. AE016752). Amino acid differences are shown in bold.

FIG. 3 illustrates a nucleic acid sequence (SEQ ID NO: 5) encoding SEQID NO: 2. The additional His-tag and carboxylamino acids encoding regionare shown in bold.

FIG. 4 illustrates a DNA sequence (SEQ ID NO: 4) encoding for SEQ ID NO:3.

FIGS. 5A and 5B illustrate a sequence comparison between different AhpFsequences from S. aureus: SEQ ID NOs: 3 (GenBank Accession No. U92441),9 (GenBank Accession No. AP004823), and 10 (GenBank Accession No.BX57183) and S. epidermidis, SEQ ID NO: 8 (GenBank Accession No.AE016752). Amino acid differences are shown in bold.

FIGS. 6A and 6B illustrate results from experiments using either a SEQID NO: 2 polypeptide (closed circles) in aluminum hydroxyphosphateadjuvant, or the adjuvant alone (triangles).

DETAILED DESCRIPTION OF THE INVENTION

The ability of SEQ ID NO: 1 related polypeptides to provide protectiveimmunity is illustrated in the Examples provided below using SEQ ID NO:2. SEQ ID NO: 2 is a derivative of SEQ ID NO: 1 containing an aminoHis-tag and three additional carboxylamino acids. The His-tagfacilitates polypeptide purification and identification.

Polypeptides structurally related to SEQ ID NO: 1 include polypeptidescontaining corresponding regions present in different S. aureus strainsand derivatives of naturally occurring regions. The amino acid sequenceof SEQ ID NO: 1 is illustrated by the bold region in FIG. 1. FIG. 1 alsoillustrates the amino His-tag present and additional carboxyl aminoacids present in SEQ ID NO: 2.

I. AhpC Sequences

S. aureus AhpC was initially identified as a protein induced by osmoticup shock having extensive similarity to E. coli alkyl hydroperoxidereductase (AhpC). (Amstrong-Buisseret et al., Microbiology141:1655-1661, 1995.) AhpC homologs of varying similarity are present inmammalian brain and in different organisms, including numerous bacterialspecies. (Chae et al., Proc. Natl. Acad. Sci. USA 91:7017-7021, 1994,Yan et al., Helicobacter 6:274-282, 2001.)

S. aureus AhpC related sequences have been given different designationsin different references. Examples of different designations are providedin TIGR(SA0452); Baba et al., Lancet 359:1819-1827, 2002 (MWO357);Kuroda et al., Lancet 357:1225-1240, 2001 (SAV0381); and Ohta et al.,DNA Research 1:51, 2004 (SAV0381 and SAV0773).

FIG. 2 provides a sequence comparison for S. aureus AhpC relatedsequences present in S. aureus (SEQ ID NO: 1) and S. epidermidis (SEQ IDNO: 6). Additional comparisons can be performed from other AhpCsequences.

Other naturally occurring AhpC sequences can be identified based on thepresence of a high degree of sequence similarity or contiguous aminoacids compared to a known AhpC sequence. Contiguous amino acids providecharacteristic tags. In different embodiments, a naturally occurringAhpC sequence is a sequence found in a Staphylococcus, preferably S.aureus, having at least 20, at least 30, or at least 50 contiguous aminoacids as in SEQ ID NO: 1; and/or having at least 85% sequence similarityor identity with SEQ ID NO: 1.

Sequence similarity can be determined by different algorithms andtechniques well known in the art. Generally, sequence similarity isdetermined by techniques aligning two sequences to obtain maximum aminoacid identity, allowing for gaps, additions and substitutions in one ofthe sequences.

Sequence similarity can be determined, for example, using a localalignment tool utilizing the program lalign (developed by Huang andMiller, Adv. Appl. Math. 12:337-357, 1991, for the <<sim>>program). Theoptions and environment variables are: —f # Penalty for the firstresidue a gap (−14 by default); —g # Penalty for each additional residuein a gap (−4 by default)-s str (SMATRIX) the filename of an alternativescoring matrix file. For protein sequences, PAM250 is used by default-w# (LINLEN) output line length for sequence alignments (60).

II. SEQ ID NO: 1 Related Polypeptides

SEQ ID NO: 1 related polypeptides contain an amino acid sequence atleast 85% identical to SEQ ID NO: 1. Reference to “polypeptide” does notprovide a minimum or maximum size limitation.

A polypeptide at least 85% identical to SEQ ID NO: 1 contains up toabout 28 amino acid alterations from SEQ ID NO: 1. Each amino acidalteration is independently either an amino acid substitution, deletion,or addition. In different embodiments, the SEQ ID NO: 1 relatedpolypeptide is at least 90%, at least 94%, or at least 99% identical toSEQ ID NO: 1; differs from SEQ ID NO: 1 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, I 1, 12, 13, 14, 15, 16, 17, 18, 19, or amino acid alterations; orconsists essentially of SEQ ID NO: 1.

An embodiment of the present invention features a polypeptide immunogencomprising or consisting essentially of a sequence at least 85%, atleast 95%, or 100% identical to amino acids 178-189 of SEQ ID NO: 1. Infurther embodiments, the polypeptide comprising amino acids 178-189 ofSEQ ID NO: 1 consists of no more than 13, 15, 20, 25, 50, 100, or 150amino acids in total; and/or the overall polypeptide is at least 85%, atleast 90%, or identical to a SEQ ID NO: 1 region and comprises aminoacids 178-189 of SEQ ID NO: 1. Additional amino acids that may bepresent include additional SEQ ID NO: 1 amino acids or other amino acidregions. A preferred additional amino acid is an amino terminusmethionine.

Reference to “consists essentially” of indicated amino acids indicatesthat the referred to amino acids are present and additional amino acidsmay be present. The additional amino acids can be at the carboxyl oramino terminus. In different embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids arepresent.

Alterations can be made to SEQ ID NO: 1 polypeptides and fragmentsthereof to obtain derivatives that induce protective immunity against S.aureus. Alterations can be performed, for example, to obtain aderivative retaining the ability to induce protective immunity againstS. aureus or to obtain a derivative that in addition to providingprotective immunity also has a region that can achieve a particularpurpose.

The sequence comparison provided in FIG. 2, and a comparison with otherS. aureus AhpC sequences, can be used to guide the design of potentialalterations. In addition, alterations can be made taking into accountknown properties of amino acids.

Generally, in substituting different amino acids to retain activity itis preferable to exchange amino acids having similar properties. Factorsthat can be taken into account for an amino acid substitution includeamino acid size, charge, polarity, and hydrophobicity. The effect ofdifferent amino acid R-groups on amino acid properties are well known inthe art. (See, for example, Ausubel, Current Protocols in MolecularBiology, John Wiley, 1987-2002, Appendix 1C.)

In exchanging amino acids to maintain activity, the replacement aminoacid should have one or more similar properties such as approximatelythe same charge and/or size and/or polarity and/or hydrophobicity. Forexample, substituting valine for leucine, arginine for lysine, andasparagine for glutamine are good candidates for not causing a change inpolypeptide functioning.

Alterations to achieve a particular purpose include those designed tofacilitate production or efficacy of the polypeptide; or cloning of theencoded nucleic acid. Polypeptide production can be facilitated throughthe use of an initiation codon (e.g., coding for methionine) suitablefor recombinant expression. The methionine may be later removed duringcellular processing. Cloning can be facilitated by, for example, theintroduction of restriction sites which can be accompanied by amino acidadditions or changes.

Efficacy of a polypeptide to induce an immune response can be enhancedthrough epitope enhancement. Epitope enhancement can be performed usingdifferent techniques such as those involving alteration of anchorresidues to improve peptide affinity for MHC molecules and thoseincreasing affinity of the peptide-MHC complex for a T-cell receptor.(Berzofsky et al., Nature Review 1:209-219, 2001.)

Preferably, the polypeptide is a purified polypeptide. A “purifiedpolypeptide” is present in an environment lacking one or more otherpolypeptides with which it is naturally associated and/or is representedby at least about 10% of the total protein present. In differentembodiments, the purified polypeptide represents at least about 50%, atleast about 75%, or at least about 95% of the total protein in a sampleor preparation.

In an embodiment, the polypeptide is “substantially purified.” Asubstantially purified polypeptide is present in an environment lackingall, or most, other polypeptides with which the polypeptide is naturallyassociated. For example, a substantially purified S. aureus polypeptideis present in an environment lacking all, or most, other S. aureuspolypeptides. An environment can be, for example, a sample orpreparation.

Reference to “purified” or “substantially purified” does not require apolypeptide to undergo any purification and may include, for example, achemically synthesized polypeptide that has not been purified.

Polypeptide stability can be enhanced by modifying the polypeptidecarboxyl or amino terminus. Examples of possible modifications includeamino terminus protecting groups such as acetyl, propyl, succinyl,benzyl, benzyloxycarbonyl or t-butyloxycarbonyl; and carboxyl terminusprotecting groups such as amide, methylamide, and ethylamide.

In an embodiment of the present invention the polypeptide immunogen ispart of an immunogen containing one or more additional regions ormoieties covalently joined to the polypeptide at the carboxyl terminusor amino terminus, where each region or moiety is independently selectedfrom a region or moiety having at least one of the following properties:enhances the immune response, facilitates purification, or facilitatespolypeptide stability. Polypeptide stability can be enhanced, forexample, using groups such as polyethylene glycol that may be present onthe amino or carboxyl terminus.

Polypeptide purification can be enhanced by adding a group to thecarboxyl or amino telininus to facilitate purification. Examples ofgroups that can be used to facilitate purification include polypeptidesproviding affinity tags. Examples of affinity tags include asix-histidine tag, trpE, glutathione and maltose-binding protein.

The ability of a polypeptide to produce an immune response can beenhanced using groups that generally enhance an immune response.Examples of groups that can be joined to a polypeptide to enhance animmune response against the polypeptide include cytokines such as IL-2.(Buchan et al., 2000. Molecular Immunology 37:545-552.)

III. AhpC-AhpF Immunogens

An AhpC-AhpF immunogen is a composition containing an AhpC and an AhpFcomponent. The AhpC component is made up of a SEQ ID NO: 1 relatedpolypeptide. The AhpF component is made up of a SEQ ID NO: 3 relatedpolypeptide.

SEQ ID NO: 1 related polypeptides contain an amino acid sequence atleast 85% identical to SEQ ID NO: 1. Different embodiments of SEQ ID NO:1 related polypeptides are described in Section II supra.

SEQ ID NO: 3 related polypeptides contain an amino acid sequence atleast 85% identical to SEQ ID NO: 3. Each amino acid alteration isindependently either an amino acid substitution, deletion, or addition.In different embodiments, the SEQ ID NO: 3 related polypeptide is atleast 90%, at least 94%, at least 99%, or identical to SEQ ID NO: 3;differs from SEQ ID NO: 3 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations; or consistsessentially of SEQ ID NO: 3.

Alterations can be made to SEQ ID NO: 3 related polypeptides to obtainderivatives used with the SEQ ID NO: 1 related polypeptide component.Alterations can be performed, for example, to obtain an overallcomposition retaining the ability to induce protective immunity againstS. aureus or to obtain an overall composition that in addition toproviding protective immunity also has a region that can achieve aparticular purpose.

Examples of different AhpF sequences that can be used to aid in thedesign of the AhpF component are provided in FIGS. 5A and 5B. Additionalguidance for producing alterations to a polypeptide are provided inSection II supra.

S. aureus AhpF related sequences have been given different designationsin different references. Examples of different designations are providedin TIGR(SA0451); Baba et al., Lancet 359:1819-1827, 2002 (MWO356);Kuroda et al., Lancet 357:1225-1240, 2001 (SAV0380 and SA0365); andEnright et al., PNAS 99:9786-9791, 2002 (SAS0357 and SAR0398).

AhpC and AhpF composition are preferably produced recombinantly usingconstructs expressing both components. For example, an E. coli straincan be engineered to coexpress ahpC and ahpF and the AhpC and AhpFcomplex can be isolated. Additional guidance and examples forpolypeptide production is provided in Section IV infra.

IV. Polypeptide Production

Polypeptides can be produced using standard techniques including thoseinvolving chemical synthesis and those involving purification from acell producing the polypeptide. Techniques for chemical synthesis ofpolypeptides are well known in the art. (See e.g., Vincent, Peptide andProtein Drug Delivery, New York, N.Y., Decker, 1990.) Techniques forrecombinant polypeptide production and purification are also well knownin the art. (See for example, Ausubel, Current Protocols in MolecularBiology, John Wiley, 1987-2002.)

Obtaining polypeptides from a cell is facilitated using recombinantnucleic acid techniques to produce the polypeptide. Recombinant nucleicacid techniques for producing a polypeptide involve introducing, orproducing, a recombinant gene encoding the polypeptide in a cell andexpressing the polypeptide.

A recombinant gene contains nucleic acid encoding a polypeptide alongwith regulatory elements for polypeptide expression. The recombinantgene can be present in a cellular genome or can be part of an expressionvector.

The regulatory elements that may be present as part of a recombinantgene include those naturally associated with the polypeptide encodingsequence and exogenous regulatory elements not naturally associated withthe polypeptide encoding sequence. Exogenous regulatory elements such asan exogenous promoter can be useful for expressing a recombinant gene ina particular host or increasing the level of expression. Generally, theregulatory elements that are present in a recombinant gene include atranscriptional promoter, a ribosome binding site, a terminator, and anoptionally present operator. A preferred element for processing ineukaryotic cells is a polyadenylation signal.

Expression of a recombinant gene in a cell is facilitated through theuse of an expression vector. Preferably, an expression vector inaddition to a recombinant gene also contains an origin of replicationfor autonomous replication in a host cell, a selectable marker, alimited number of useful restriction enzyme sites, and a potential forhigh copy number. Examples of expression vectors are cloning vectors,modified cloning vectors, specifically designed plasmids and viruses.

Due to the degeneracy of the genetic code, a large number of differentencoding nucleic acid sequences can be used to code for a particularpolypeptide. The degeneracy of the genetic code arises because almostall amino acids are encoded by different combinations of nucleotidetriplets or “codons”. Amino acids are encoded by codons as follows:

A=Ala=Alanine: codons GCA, GCC, GCG, GCUC=Cys=Cysteine: codons UGC, UGUD=Asp=Aspartic acid: codons GAC, GAUE=Glu=Glutamic acid: codons GAA, GAGF=Phe=Phenylalanine: codons UUC, UUUG=Gly=Glycine: codons GGA, GGC, GGG, GGUH=His=Histidine: codons CAC, CAUI=Ile=Isoleucine: codons AUA, AUC, AUUK=Lys=Lysine: codons AAA, AAGL=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUUM=Met=Methionine: codon AUGN=Asn=Asparagine: codons AAC, AAUP=Pro=Proline: codons CCA, CCC, CCG, CCUQ=Gln=Glutamine: codons CAA, CAGR=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGUS=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCUT=Thr=Threonine: codons ACA, ACC, ACG, ACUV=Val=Valine: codons GUA, GUC, GUG, GUUW=Trp=Tryptophan: codon UGGY=Tyr=Tyrosine: codons UAC, UAU

Suitable cells for recombinant nucleic acid expression of SEQ ID NO: 1or SEQ ID NO: 3 related polypeptides are prokaryotes and eukaryotes.Examples of prokaryotic cells include E. coli; members of theStaphylococcus genus, such as S. aureus; members of the Lactobacillusgenus, such as L. plantarum; members of the Lactococcus genus, such asL. lactis; and members of the Bacillus genus, such as B. subtilis.Examples of eukaryotic cells include mammalian cells; insect cells;yeast cells such as members of the Saccharomyces genus (e.g., S.cerevisiae), members of the Pichia genus (e.g., P. pastoris), members ofthe Hansenula genus (e.g., H. polymorpha), members of the Kluyveromycesgenus (e.g., K lactis or K fragilis) and members of theSchizosaccharomyces genus (e.g., S. pombe). Techniques for recombinantgene production, introduction into a cell, and recombinant geneexpression are well known in the art. Examples of such techniques areprovided in references such as Ausubel, Current Protocols in MolecularBiology, John Wiley, 1987-2002, and Sambrook et al., Molecular Cloning,A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor LaboratoryPress, 1989.

If desired, expression in a particular host can be enhanced throughcodon optimization. Codon optimization includes use of more preferredcodons. Techniques for codon optimization in different hosts are wellknown in the art.

Polypeptides may contain post translational modifications, for example,N-linked glycosylation, O-linked glycosylation, or acetylation.Reference to “polypeptide” or an “amino acid” sequence of a polypeptideincludes polypeptides containing one or more amino acids having astructure of a post-translational modification from a host cell, such asa yeast host.

Post translational modifications can be produced chemically or by makinguse of suitable hosts. For example, in S. cerevisiae the nature of thepenultimate amino acid appears to determine whether the N-terminalmethionine is removed. Furthermore, the nature of the penultimate aminoacid also determines whether the N-terminal amino acid isN^(α)-acetylated (Huang et al., Biochemistry 26: 8242-8246, 1987).Another example includes a polypeptide targeted for secretion due to thepresence of a secretory leader (e.g., signal peptide), where the proteinis modified by N-linked or O-linked glycosylation. (Kukuruzinska et al.,Ann. Rev. Biochem. 56:915-944, 1987.)

V. Adjuvants

Adjuvants are substances that can assist an immunogen in producing animmune response. Adjuvants can function by different mechanisms such asone or more of the following: increasing the antigen biologic orimmunologic half-life; improving antigen delivery to antigen-presentingcells; improving antigen processing and presentation byantigen-presenting cells; and inducing production of immunomodulatorycytokines. (Vogel, Clinical Infectious Diseases 30 (suppl. 3):S266-270,2000.)

A variety of different types of adjuvants can be employed to assist inthe production of an immune response. Examples of particular adjuvantsinclude aluminum hydroxide, aluminum phosphate, or other salts ofaluminum, calcium phosphate, DNA CpG motifs, monophosphoryl lipid A,cholera toxin, E. coli heat-labile toxin, pertussis toxin, muramyldipeptide, Freund's incomplete adjuvant, MF59, SAF, immunostimulatorycomplexes, liposomes, biodegradable microspheres, saponins, nonionicblock copolymers, muramyl peptide analogues, polyphosphazene, syntheticpolynucleotides, IFN-γ, IL-2, IL-12, and ISCOMS. (Vogel ClinicalInfectious Diseases 30 (suppl 3):S266-270, 2000, Klein et al., Journalof Pharmaceutical Sciences 89:311-321, 2000, Rimmelzwaan et al., Vaccine19:1180-1187, 2001, Kersten Vaccine 21:915-920, 2003, O'Hagen Curr. DrugTarget Infect. Disord., 1:273-286, 2001.)

VI. Patients

A “patient” refers to a mammal capable of being infected with S. aureus.A patient can be treated prophylactically or therapeutically.Prophylactic treatment provides sufficient protective immunity to reducethe likelihood, or severity, of a S. aureus infection. Therapeutictreatment can be performed to reduce the severity of a S. aureusinfection.

Prophylactic treatment can be performed using a vaccine containing animmunogen described herein. Such treatment is preferably performed on ahuman. Vaccines can be administered to the general population or tothose persons at an increased risk of S. aureus infection.

Persons with an increased risk of S. aureus infection include healthcare workers; hospital patients; patients with a weakened immune system;patients undergoing surgery; patients receiving foreign body implants,such as a catheter or a vascular device; patients facing therapy leadingto a weakened immunity; and persons in professions having an increasedrisk of burn or wound injury. (The Staphylococci in Human Disease,Crossley and Archer (ed.), Churchill Livingstone Inc. 1997.)

Non-human patients that can be infected with S. aureus include cows,pigs, sheep, goats, rabbits, horses, dogs, cats, monkeys, rats, andmice. Treatment of non-human patients is useful in protecting pets andlivestock, and in evaluating the efficacy of a particular treatment.

VII. Combination Vaccines

Immunogens described herein can be used alone, or in combination withother immunogens, to induce an immune response. Additional immunogensthat may be present include: one or more additional S. aureusimmunogens, such as those referenced in the Background of the Inventionsupra; one or more immunogens targeting one or more other Staphylococcusorganisms such as S. epidermidis, S. haemolyticus, S. warneri, or S.lugunensis; and one or more immunogens targeting other infectionsorganisms.

VIII. Animal Model System

An animal model system was used to evaluate the efficacy of an immunogento produce a protective immune response against S. aureus. The animalmodel was a slow kinetics lethality model involving S. aureus preparedfrom cells in stationary phase, appropriately titrated, andintravenously administered. This slow kinetics of death providessufficient time for the specific immune defense to fight off thebacterial infection (e.g., 10 days rather 24 hours).

S. aureus cells in stationary phase can be obtained from cells grown onsolid medium. They can also be obtained from liquid, however the resultswith cells grown on solid medium were more reproducible. Cells canconveniently be grown overnight on solid medium. For example, S. aureuscan be grown from about 18 to about 24 hours under conditions where thedoubling time is about 20-30 minutes.

S. aureus can be isolated from solid or liquid medium using standardtechniques to maintain S. aureus potency. Isolated S. aureus can bestored, for example, at −70° C. as a washed high density suspension(>10⁹ colony forming units (CFU)/mL) in phosphate buffered salinecontaining glycerol.

The S. aureus challenge should have a potency providing about 80 to 90%death in an animal model over a period of about 7 to 10 days starting onthe first or second day. Titration experiments can be performed usinganimal models to monitor the potency of the stored

S. aureus inoculum. The titration experiments can be performed about oneto two weeks prior to an inoculation experiment.

IX. Antibodies

Immunogens containing SEQ ID NO: 1 related polypeptides and AhpC-AhpFcompositions can be used to produce isolated binding proteins that bindto the immunogen or to S. aureus. Such binding proteins have differentuses including use in polypeptide purification, S. aureusidentification, or in therapeutic or prophylactic treatment against S.aureus infection. Preferably, the binding protein is substantially freeof serum proteins.

A binding protein comprises a first variable region and a secondvariable region. The variable regions have the structure of an antibodyvariable region from a heavy or light chain. Antibody heavy and lightchain variable regions contain three complementary determining regionsinterspaced onto a framework. The complementary determining regions areprimarily responsible for recognizing a particular epitope. Examples ofantibody binding protein include single-chain antibodies, a completeantibody, an antibody fragment, and derivatives thereof.

A preferred antigen binding protein is a monoclonal antibody. Referenceto a “monoclonal antibody” indicates a collection of antibodies havingthe same, or substantially the same, complementary determining regionand binding specificity. The variation in the monoclonal antibodies isthat which would occur if the antibodies were produced from the sameconstruct(s).

Monoclonal antibodies can be produced, for example, from a particularhybridoma and from a recombinant cell containing one or more recombinantgenes encoding the antibody. The antibody may be encoded by more thanone recombinant gene where, for example, one gene encodes the heavychain and one gene encodes the light chain.

Antibody fragments containing an antibody variable region include Fv,Fab, and Fab₂ regions. Each Fab region contains a light chain made up ofa variable region and a constant region, and a heavy chain regioncontaining a variable region and a constant region. A light chain isjoined to a heavy chain by disulfide bonding through constant regions.The light and heavy chain variable regions of a Fab region provide foran Fv region that participates in antigen binding.

The antibody variable region can also be part of protein containingvariable regions such as single chain antibody and a minibody. A singlechain antibody contains a light and a heavy variable region joinedtogether by a linker. The linker can be, for example, about 5 to 16amino acids. A minibody is a single chain-CH₃ fusion protein that selfassembles into a bivalent dimer of about 80 kDa.

Specificity of the variable region is determined by three hypervariableregions (also referred to as complementarity determining regions), thatare interposed between more conserved flanking regions (also referred toas framework regions). Amino acids associated with framework regions andcomplementarity deteanining regions can be numbered and aligned asdescribed by Kabat et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1991.

Techniques for generating antigen binding protein such as a single-chainantibody, an antibody, or an antibody fragment are well known in theart. Examples of such techniques include the use of phage displaytechnology, identification and humanization of rodent antibodies, andgeneration of human antibodies using a XenoMouse or Trans-Chromo mouse.(E.g., Azzazy et al., Clinical Biochemistry 35:425-445, 2002, Berger etal., Am. J. Med. Sci. 324(1):14-40, 2002.)

Murine antibodies can be humanized, and CDR's, can be grafted on tohuman antibody frameworks using techniques well known in art. Suchtechniques are generally described with reference to humanizing murineantibodies by grafting murine variable regions onto a human antibodyframework and, if needed making further modifications. (E.g., O'Brien etal., Humanization of Monoclonal Antibodies by CDR Grafting, p 81-100,From Methods in Molecular Biology Vol 207: Recombinant antibodies forCancer Therapy. Methods and Protocols (Eds. Welschof and Krauss) HumanaPress, Totowa, N.J., 2003.)

Antigen binding protein are preferably produced using recombinantnucleic acid techniques or through the use of a hybridoma. Recombinantnucleic acid techniques involve constructing a nucleic acid template forprotein synthesis. A hybridoma is an immortalized cell line producingthe antigen binding protein.

Recombinant nucleic acid encoding an antigen binding protein can beexpressed in a host cell that in effect serves as a factory for theencoded protein. The recombinant nucleic acid can provide a recombinantgene encoding the antigen binding protein that exists autonomously froma host cell genome or as part of the host cell genome.

A recombinant gene contains nucleic acid encoding a protein along withregulatory elements for protein expression. Generally, the regulatoryelements that are present in a recombinant gene include atranscriptional promoter, a ribosome binding site, a terminator, and anoptionally present operator. A preferred element for processing ineukaryotic cells is a polyadenylation signal. Antibody associatedintrons may also be present. Examples of expression cassettes forantibody or antibody fragment production are well known in art. (E.g.,Persic et al., Gene 187:9-18, 1997, Boel et al., J. Immunol. Methods239:153-166, 2000, Liang et al., J. Immunol. Methods 247:119-130, 2001.)

Expression of a recombinant gene in a cell is facilitated using anexpression vector. Preferably, an expression vector, in addition to arecombinant gene, also contains an origin of replication for autonomousreplication in a host cell, a selectable marker, a limited number ofuseful restriction enzyme sites, and a potential for high copy number.Examples of expression vectors for antibody and antibody fragmentproduction are well known in art. (E.g., Persic et al., Gene 187:9-18,1997, Boel et al., J. Immunol. Methods 239:153-166, 2000, Liang et al.,J. Immunol. Methods 247:119-130, 2001.)

If desired, nucleic acid encoding an antibody may be integrated into thehost chromosome using techniques well known in the art. (See, Ausubel,Current Protocols in Molecular Biology, John Wiley, 1987-1998, Mark etal., U.S. Pat. No. 6,743,622.)

A variety of different cell lines can be used for recombinant antigenbinding protein expression, including those from prokaryotic organisms(e.g., E. coli, Bacilli, and Streptomyces) and from Eukaryotic (e.g.,yeast, Baculovirus, and mammalian). (Breitling et al., RecombinantAntibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag,1999.)

Preferred hosts for recombinant antigen binding protein expression aremammalian cells able to produce antigen binding protein with proper posttranslational modifications. Post translational modifications includedisulfide bond formation and glycosylation. Another type of posttranslational modification is signal peptide cleavage.

Proper glycosylation can be important for antibody function. (Yoo etal., Journal of Immunological Methods 261:1-20, 2002.) Naturallyoccurring antibodies contain at least one N-linked carbohydrate attachedto a heavy chain. (Id.) Additional N-linked carbohydrates and O-linkedcarbohydrates may be present and may be important for antibody function.(Id.)

Different types of mammalian host cells can be used to provide forefficient post-translational modifications. Examples of such host cellsinclude Chinese hamster ovary (CHO), HeLa, C6, PC 12, and myeloma cells.(Yoo et al., Journal of Immunological Methods 261:1-20, 2002, Persic etal., Gene 187:9-18, 1997.)

A hybridoma can be produced using techniques such as those described inAusubel Current Protocols in Molecular Biology, John Wiley, 1987-1998,Harlow et al., Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, 1988, and Kohler et al., Nature 256, 495-497, 1975.

X. Administration

Immunogens and binding protein can be formulated and administered to apatient using the guidance provided herein along with techniques wellknown in the art. Guidelines for pharmaceutical administration ingeneral are provided in, for example, Vaccines Eds. Plotkin andOrenstein, W.B. Sanders Company, 1999; Remington's PharmaceuticalSciences 20^(th) Edition, Ed. Gennaro, Mack Publishing, 2000; and ModernPharmaceutics 2^(nd) Edition, Eds. Banker and Rhodes, Marcel Dekker,Inc., 1990, each of which are hereby incorporated by reference herein.

Pharmaceutically acceptable carriers facilitate storage andadministration of an immunogen to a patient. Pharmaceutically acceptablecarriers may contain different components such as a buffer, sterilewater for injection, normal saline or phosphate buffered saline,sucrose, histidine, salts and polysorbate.

Immunogens and binding protein can be administered by different routessuch as subcutaneous, intramuscular, or mucosal. Subcutaneous andintramuscular administration can be performed using, for example,needles or jet-injectors.

Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including age, weight, sex and medicalcondition of the patient; the route of administration; the desiredeffect; and the particular compound employed. The immunogen or bindingprotein can be used in multi-dose formats. It is expected that a dosewould consist of the range of 1.0 μg to 1.0 mg total polypeptide, indifferent embodiments of the present invention the range is 0.01 mg to1.0 mg and 0.1 mg to 1.0 mg.

The timing of doses depends upon factors well known in the art. Afterthe initial administration one or more booster doses may subsequently beadministered to maintain or boost antibody titers. An example of adosing regime would be day 1, 1 month, a third dose at either 4, 6 or 12months, and additional booster doses at distant times as needed.

EXAMPLES

Examples are provided below further illustrating different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Protective Immunity

This example illustrates the ability of SEQ ID NO: 1 relatedpolypeptides to provide protective immunity in an animal model. SEQ IDNO: 2, a His-tagged derivative of SEQ ID NO: 1, was used to provideprotective immunity.

SEQ ID NO: 2 Cloning and Expression

The protein encoded by COL SA0452 was designed to be expressed from thepET16b vector (EMD Biosciences, Madison, Wis.) with the N-terminalhistidine residues and the stop codon encoded by the vector. Alsoencoded by the vector are nine additional amino acids after thehistidine tag and three additional amino acids at the carboxyl terminus.PCR primers were designed to amplify COL SA0452 starting at the firstserine codon and ending prior to the stop codon at the terminalisoleucine residue. The forward and reverse primers were:5′GGGAATTCCATATGTCATTAATTAACAAAGAAATCTTACC3′ (SEQ ID NO: 7) and 5′GGGCTCAGCGATITTACCTACTAAATCTAAACCAG3′ (SEQ ID NO: 11), respectively andcontained additional restriction sites (underlined), i.e., NdeI (forwardprimer) and Bpu1102II (reverse primer) and a GC clamp to facilitatecloning into the expression vector.

Genomic DNA was purified from S. aureus COL strain MB5393 and used astemplate for PCR. A 50-mL culture was grown overnight in DIFCO TrypticSoy Broth (Becton Dickinson, Sparks, Md.) at 37° C. and the cells werecollected by centrifugation. The cells were washed once in 5 mL of 10 mMTris pH 7.5, 25% sucrose and resuspended in the same solution containing50 μg/mL lysostaphin (Sigma, St. Louis, Mo.). The mixture was incubatedat 37° C. for 1 hour and subsequently, 2.5 mL of 0.25 M EDTA pH 8.0 wasadded. After incubation for 15 minutes on ice, 7.5 mL 2% sarkosyl wasadded and the mixture was swirled gently and allowed to sit for 30 moreminutes on ice. One-hundred fifty μL RNAse, 5 mg/mL in 0.1 M sodiumacetate, was added and the mixture was incubated for one hour at 37° C.Next, 0.6 mL proteinase K, 25 mg/mL, was added and the incubation wascontinued for 2 hours followed by an overnight incubation at 4° C. Thelysate was extracted with 15 mL water-saturated phenol by gentlerotation for 30 minutes at room temperature.

To separate phases the mixture was centrifuged at 4,000 rpm for 10minutes at room temperature. The aqueous phase was collected and thephenol extraction was repeated two more times. Next, the aqueous phasewas extracted ten times with an equal volume of chloroform. After thelast extraction, the aqueous phase was collected, the volume wasmeasured, and one half volume 7.5 M ammonium acetate was added.

The DNA was precipitated by adding two volumes of 100% ethanol andcollected by spooling with a glass rod. The DNA was dissolved in 5.0 mLTE containing 4 μL diethylpyrocarbonate overnight at 4° C. The ethanolprecipitations were repeated two more times.

The ahpC gene was amplified by PCR in a 50 μL volume reaction preparedin duplicate. Each contained 250 ng genomic DNA, 125 ng each forward andreverse primer, 1 microliter 10 mM dNTPs, 2.5 units of native Pfupolymerase and 1×Pfu buffer (Stratagene, La. Jolla Calif.). Thethermacycling conditions were as follows: one cycle of 94° C. for 5minutes; 30 cycles of 94° C. for 45 seconds, 56° C. for 45 seconds, 72°C. for one minute; one cycle of 72° C. for 10 minutes. The amplified DNAsequence (584 bp) was digested with the appropriate restriction enzymesand gel-purified using GENE CLEAN II® (QBIOgene, Carlsbad, Calif.)according to the manufacturer's directions. The DNA was ligated into thepET16b vector using the NdeI/Bpu1102I sites that had been engineeredinto the PCR primers and introduced into E. coli NovaBlue competentcells (EMD Biosciences).

The transformation mixture was grown overnight at 37° C. on low saltLennox L Broth agar plates containing 100 μg/mL each ampicillin, IPTG,and X-gal, prepared using IMMEDIA™ Amp Agar (Invitrogen, Carlsbad,Calif.), according to the manufacturer's instructions. Colonies wereselected and grown in Luria Broth (LB) with 50 μg/mL ampicillin, DNAminipreps were made (Promega), and the appropriate insert was determinedby restriction endonuclease digestion. The plasmid DNA from twominipreps was sequenced, and a clone containing no DNA changes from thedesired sequence was selected and designated pAhpC5.

E. coli BLR (DE3) competent cells (EMD Biosciences) were transformedwith pAhpC5 and grown on LB plates containing ampicillin (100 μg/mL). Totest for expression of ahpC, an isolated colony was inoculated into 5 mLof liquid LB (ampicillin) and incubated at 37° C., 220 rpm for 6 hours.The culture was held overnight at 4° C. and inoculated the next day into20.0 mL LB broth (ampicillin) such that the starting OD₆₀₀ equaled 0.02.The culture was incubated at 37° C., 220 rpm for four hours to anOD₆₀₀=0.8. Induction of expression was compared at three differenttemperatures. Forty-five microliters of 100 mM IPTG was added to three4.5 mL culture volumes (final IPTG concentration of 1 mM) and incubatedat 37° C. for 3 hours, and 25° C. or 18° C. for 24 hours, all withshaking at 220 rpm. For lysate preparation, 1.5 mL and 1.0 mL culturevolume from uninduced and induced cultures, respectively, were collectedby centrifugation and resuspended in 300 μl, of BUGBUSTER HT (EMDSciences) and 3 μL Proteinase Inhibitor Cocktail (Sigma, St. Louis,Mo.). The mixtures were held on ice for 5 minutes and subsequentlysonicated three times for ten seconds each with cooling in between. Toobtain “soluble” and “insoluble” fractions the mixture was centrifugedat 14,000 rpm for five minutes at 4° C. The supernatant was designated“soluble” and the pellet was resuspended in 300 μL of BUGBUSTER HT and 3μL Proteinase Inhibitor Cocktail and designated “insoluble”. Proteinconcentration was determined by the BIO-RAD Protein Assay Dye Reagentsystem (BIO-RAD, Hercules, Calif.) according to the manufacturer'sinstructions.

For analysis of expression of ahpC (encoded by SEQ ID NO: 3) byCOOMASSIE staining of SDS-PAGE gels, samples were subjected toelectrophoresis on 4-15% gradient Tris-HC₁-Criterion gels (BIO-RAD) in1× Tris glycine SDS buffer (BIO-RAD) under reducing and denaturingconditions. To estimate protein size, standards between 15 and 250 kDa(BIO-RAD) were run in parallel with the lysates. The gels were stainedwith BIO-SAFE COOMASSIE, a COOMASSIE G250 stain (BIO-RAD) according tothe manufacturer's protocol.

A 24-kDa protein was specifically detected in lysates prepared fromsamples induced at all three temperature. Good expression was obtainedat all three temperatures with ahpC localizing to both the soluble andinsoluble fraction. Induction at 25° C. was the optimal temperature forproducing soluble ahpC.

SEQ ID NO: 2 Purification

Direct scale-up of the above small scale procedure into stirred tankfermenters (30 liter scale) with a 20 liter working volume was achieved.Inoculum was cultivated in a 250 mL flask containing 50 mL ofLuria-Bertani (LB) medium (plus ampicillin) and inoculated with 1 mL offrozen seed culture and cultivated for 6 hours. One mL of this seed wasused to inoculate a 2 liter flask containing 500 mL of LB medium (plusampicillin) and incubated for 16 hours. A large scale fermenter (30liter scale) was cultivated with 20 liters of LB medium (plusampicillin). The fermentation parameters of the fermenter were:pressure=5 psig, agitation speed=300 rpms, airflow=7.5 liters/minute andtemperature=37° C. Cells were incubated to an optical density (OD) of1.3 optical density units, at a wavelength of 600 nm, and induced withIsopropyl-β-K-Thiogalactoside (IPTG) at a concentration of 1 mM.Induction time with IPTG was two hours. Cells were harvested by loweringthe temperature to 15° C., concentrated by passage through a 500KMWCOhollow fiber cartridge, and centrifuged at 8,000 times gravity at 4° C.for 20 minutes. Supernatants were decanted and the recombinant E. coliwet cell pellets were frozen at −70° C.

Frozen recombinant E. coli cell paste (24 grams) was thawed andresuspended in two volumes of Lysis Buffer (50 mM sodium phosphate, pH8.0, 0.15 M NaCl, 2 mM magnesium chloride, 10 mM imidazole, 20 mM2-mercaptoethanol, 0.1% Tween-80, and protease inhibitor cocktail(COMPLETE™, EDTA-Free, Roche # 1873580-one tablet per 50 mL LysisBuffer). Benzonase (EM #1.01697.0002) was added to the cell suspensionat 125 Units/mL). A lysate was prepared with a microfluidizer. TheLysate was stirred for three hours at 4° C., and was clarified bycentrifugation at 10,000×g for 10 minutes at 4° C. The supernatant wasfiltered through a glass-fiber pre-filter Millipore and NaCl was addedto a final concentration of 0.5 M from a 5 M stock solution. TheFiltered Supernatant was added to Ni-NTA agarose chromatography resin(Qiagen #30250) and the slurry was mixed overnight at 4° C. The slurryof chromatography resin was poured into a chromatography column and thenon-bound fraction was collected by gravity from the column outlet. Thecolumn was washed with ten column volumes of Wash Buffer (50 mM sodiumphosphate, pH 8.0, 0.5 M NaCl, 2 mM magnesium chloride, 10 mM imidazole,20 mM 2-mercaptoethanol, 0.1% Tween-80, and protease inhibitor cocktail(COMPLETE™, EDTA-Free, Roche # 1873580-one tablet per 50 mL WashBuffer). The column was eluted with Elution Buffer (50 mM sodiumphosphate, pH 7.4, 0.3 M imidazole, 2 mM magnesium chloride, 0.1%Tween-80, and 20 mM 2-mercaptoethanol). Fractions containing proteinwere identified by dot blot on nitrocellulose membrane with Ponceau-Sstaining, and fractions containing the highest protein concentrationswere pooled to make the Ni-IMAC Product. The Ni-IMAC Product wasfractionated by SEC. SEC fractions containing the product protein wereidentified by SDS/PAGE with COOMASSIE staining. Product-containing SECfractions were pooled to make the SEC Product. The SEC Product wassterile-filtered and adsorbed on aluminum hydroxyphosphate adjuvant at afinal concentration of 0.2 mg/mL.

Preparation of S. aureus Challenge

S. aureus was grown on TSA plates at 37° C. overnight. The bacteria werewashed from the TSA plates by adding 5 mL of PBS onto a plate and gentlyresuspending the bacteria with a sterile spreader. The bacterialsuspension was spun at 6000 rpm for 20 minutes using a SORVALL RC-5Bcentrifuge (DuPont Instruments). The pellet was resuspended in 16%glycerol and aliquots were stored frozen at −70° C.

Prior to use, inocula were thawed, appropriately diluted and used forinfection. Each stock was titrated at least 3 times to determine theappropriate dose inducing slow kinetics of death in naive mice. Thepotency of the bacterial inoculum (80 to 90% lethality) was constantlymonitored to assure reproducibility of the model. Ten days before eachchallenge experiment, a group of 10 control animals (immunized withadjuvant alone) were challenged and monitored.

Protection Studies for a SEQ ID NO: 2 Polypeptide

In two independent experiments, twenty BALB/c mice each were immunizedwith three doses of SEQ ID NO: 2 polypeptide (20 μg per injection) onaluminum hydroxyphosphate adjuvant (450 μg per injection), and 20 miceeach were injected with Aluminum hydroxyphosphate adjuvant (450 μg perinjection). Aluminum hydroxyphosphate adjuvant (AHP) is described byKlein et al., Journal of Pharmaceutical Sciences 89: 311-321, 2000. Thematerials were administered as two 50 μL intramuscular injections ondays 0, 7 and 21. The mice were bled on day 28, and their sera werescreened by ELISA for reactivity to SEQ ID NO: 2.

On day 35 of each experiment the mice were challenged by intravenousinjection of S. aureus (dose 7×10⁸ CFU/mL). The mice were monitored overa 11 day period for survival. At the end of the first experiment 12 micesurvived in the SEQ ID NO: 2 polypeptide immunized group, compared to 5surviving in the AHP control group. The results are illustrated in FIG.6A. In the second experiment 11 mice survived in the SEQ ID NO: 2polypeptide immunized group, compared to 7 surviving in the AHP controlgroup. The results are illustrated in FIG. 6B.

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1-9. (canceled)
 10. An isolated nucleic acid comprising a recombinantgene comprising a sequence of nucleotides that encodes a polypeptideimmunogen, wherein the polypeptide immunogen comprises an amino acidsequence with up to 9 amino acid alterations from SEQ ID NO:1, whereinthe amino acid sequence is not SEQ ID NO:1, and wherein the polypeptideimmunogen provides protective immunity against S. aureus.
 11. Anexpression vector comprising the nucleic acid of claim
 10. 12. Arecombinant cell comprising the expression vector of claim
 11. 13. Amethod of making a S. aureus polypeptide that provides protectiveimmunity comprising the steps of: (a) growing the recombinant cell ofclaim 12 under conditions wherein said polypeptide is expressed; and (b)purifying said polypeptide. 14-20. (canceled)
 21. The isolated nucleicacid of claim 10, wherein the polypeptide immunogen consists of aminoacids 22-213 of SEQ ID NO:2.
 22. The isolated nucleic acid of claim 10,wherein the polypeptide immunogen comprises an amino acid sequence thatdiffers from SEQ ID NO:1 by up to 5 amino acid alterations.
 23. Theisolated nucleic acid of claim 10, wherein the polypeptide immunogencomprises an amino acid sequence that differs from SEQ ID NO:1 by up to3 amino acid alterations.